J. Biol. Chem., Vol. 262, Issue 34, 16305-16313, 12, 1987

Diacylglycerol metabolism in mast cells. Analysis of lipid metabolic pathways using molecular species analysis of intermediates

DA Kennerly
Department of Internal Medicine, University of Texas Health Science Center, Dallas 75235.

These studies assess the metabolic source and fate of cellular 1,2- diacylglycerol (DAG), an intermediate that increases with physiologic stimulation, participates in the regulation of protein phosphorylation, and acts as a substrate for arachidonic acid release. The quantitation of the molecular species of DAG and one of its metabolic products, phosphatidic acid (PA), was assessed in the purified rat mast cell, a model system with marked quantitative constraints but with rapid and extensive secretion after receptor stimulation. Cellular DAG was extracted, partially purified, radioactively phosphorylated to form [32P]PA, and, after conversion to its dimethyl phosphoric acid ester, molecular species separations were undertaken using reversed phase HPLC and/or argentation TLC. Quantitation of 0.5 pmol of a single molecular species of cellular DAG was achieved, but HPLC was not alone sufficient to resolve all molecular species of interest. More importantly, comparison of mast cell DAG with [32P]PA generated in 32Pi-prelabeled cells revealed that the sub-classes that contained arachidonic acid species represent only 11% of the total DAG, while that of [32P]PA was 41% in resting cells. [32P]PA and, to a variable extent, DAG showed preferential increases in arachidonate-containing subclasses after stimulation (to 50.9 and 13.9%, respectively). These data suggest that a large portion of the increased mass of DAG seen during stimulation was probably not derived by phosphoinositide hydrolysis. This type of molecular species analysis of intermediates of important phospholipid metabolic pathways should help to establish the metabolic origin and fate of these and other compounds.
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